Stability and catalytic properties of subtilisin in acetonitrile/dimethylformamide mixtures with low water content

Subtilisin 72 suspension retained high activity and stability in the triple mixtures acetonitrile/DMF/water (DMF concentration was up to 70% (v/v)). The synthetic activity of subtilisin suspension was investigated using the model reaction of tetrapeptide Z-Ala-Ala-Leu-Phe-pNA formation from Z-Ala-Ala-Leu-OMe and Phe-pNA ([S]=30 mM, [E]=6 μM; [S]/[E] molar ratio 5000:1). In the systems containing up to 60% DMF the 95% product yield was reached within 2 h. With DMF concentration increasing to 95%, the subtilisin catalytic efficiency notably decreased, though the product yield was still 30%. In the mixture acetonitrile/80%DMF/water, the effects of enzyme concentration, the reaction time and water content on the reaction progress were studied.